EP0048497A2 - Vecteur de transduction de DNA et micro-organisme le contenant - Google Patents

Vecteur de transduction de DNA et micro-organisme le contenant Download PDF

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Publication number
EP0048497A2
EP0048497A2 EP81107594A EP81107594A EP0048497A2 EP 0048497 A2 EP0048497 A2 EP 0048497A2 EP 81107594 A EP81107594 A EP 81107594A EP 81107594 A EP81107594 A EP 81107594A EP 0048497 A2 EP0048497 A2 EP 0048497A2
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Prior art keywords
dna
fragment
promoter
recombinant
vector
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EP81107594A
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German (de)
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EP0048497A3 (fr
Inventor
Seiga Itoh
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KH Neochem Co Ltd
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Kyowa Hakko Kogyo Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

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  • the present invention relates generally to a novel DNA transducing vector and a novel recombinant DNA, and more specifically to a DNA transducing vector carrying a P 1 promoter derived from ribosomal RNA of E scherichia coli and a recombinant DNA carrying the P 1 promoter.
  • D N A transducing vectors and recombinant D N As are useful in recombinant technology for cloning genes of animals, plants and microorganisms and the expression thereof.
  • transducing vectors One of the vectors which is most frequently used for the expression of the cloned gene for proteins derived from animals in Escherichia coli is a transducing vector carrying a lactose promoter.
  • Lactose operon the operon for the lactose- utilizing system in Escherichia coli, is one of the well known operons.
  • Three genes which encode ⁇ -galactosidase, galactoside permiase and galactoside acetyltransferase are aligned downstream of the lactose promoter and are expressed under the control of the promoter.
  • the ribosome of E. coli is a complex (70S, M .W.: 2.8 x 10 6 dalton) of three RNAs (16S, 23S and 5S) and 55 proteins.
  • the ribosome is an important intracellular particle which effects the synthesis of proteins and is composed of two particles, 30S and 50S. More precisely, one particle (30S) is a ribonucleoprotein composed of an RNA (16S, M.W.: 5.6 x 10 dalton) and 21 proteins and the other particle (50S) is that composed of two RNAs (23S, M.W.: 1.1 x 10 6 dalton, SS, M . W .: 4 x 10 4 dalton) and 34 proteins.
  • Ribosomes play the most important role in the translation process.
  • ribosomes are generally present in one cell of E. coli but the amount thereof varies depending on the growth rate of the microorganism. That is, the amount of ribosome is proportional to the growth rate of the microorganism. It is known that the growth rate of E. coli in a nutrient-rich medium is high, i.e. about one division per 20 minutes and under such condition ribosomal RNAs increase in proportion to the growth rate of the microorganism [N. O. Kjeldgaard, J. Mol. Biol, 6,341 (1963)].
  • Ribosomal RNA genes of E. coli are lined in the order of two promoters P 1 and P 2 , 16S RNA, 23S RNA and 5S RNA. The synthesis of the three RNAs are under the control of the promoters P 1 and P 2 .
  • Such ribosomal RNA genes (operon) of E. coli are present in the ratio of 7 to one chromosome and are called ribosomal RNA gene (rrn) A, B, C, D, E, F and G, respectively [M. Nomura: Ann. Rev. Genet., 11, 297 - 347 (1977)].
  • tandem promoters named P 1 and P 2 are present, that is, P 1 and P 2 are located at about 300 base pairs and about 200 base pairs upstream from the 5' end of the structural gene 16S RNA, respectively.
  • a DNA transducing vector and a recombinant DNA carrying the P 1 promoter has been made and it has been found that the P 1 promoter has a stronger promoter activity than that of the lactose promoter.
  • P 1 promoter derived from ribosomal RNA of Escherichia coli (referred to as "P 1 fragment", hereinafter) has a stronger activity than the lactose promoter. Therefore, a DNA transducing vector or a recombinant DNA containing the P 1 promoter is useful for cloning genes of animals, plants and microorganisms and also the expression thereof.
  • the P 1 fragment is prepared from the transducing phage DNA, ⁇ met A 20.
  • Ribosomal RNA gene is cloned in the transducing phage DNA and ribosomal RNA promoter is included therein.
  • the phage DNA is digested with. a restriction enzyme and the desired P 1 fragment is purified by agarose gel electrophoresis or polyacrylamide gel electrophoresis.
  • the P 1 fragment is inserted into a plasmid vector pBR 325.
  • the above processes are carried out according to the methods used generally in recombinant technology. That is, the pBR 325 is digested with a restriction enzyme such as Eco RI, Hind III and Barn HI and the P 1 fragment is combined with the digested pBR 325 using T4DNA ligase. As is illustrated in Flow Sheet I, the P 1 fragment has Eco RI and Hind III restriction sites at both ends.
  • the Hind II site of the P 1 fragment is changed to a Hind III site with the Hind III linker prior to the insertion.
  • the recombinant plasmid of P 1 fragment and pBR 325 is introduced into an E. coli strain and the transformant, grown on an agar medium containing 1 to 100 ⁇ g/ml, preferably about 20 ⁇ g/:ml of ampicillin, is readily selected for tetracycline resistance and chloramphenicol sensitivity.
  • the construction of the recombinant plasmid is carried out under the following conditions. Digestion of DNA with restriction enzymes is carried out as follows. Generally 0.1 to 20 ⁇ g of DNA is digested with 0.1 to 100 units, preferably 1 to 3 units of a restriction enzyme per 1 ⁇ g of DNA in the presence of 2 to 200 mM, preferably 10 to 40 mM Tris-HCl (pH 6.0 to 9.5, preferably pH 7.0 to 8.0), 2 to 100 mM NaCl and 2 to 20 mM, preferably 5 to 10 mM MgCl 2 at a temperature of 18 to 42°C, preferably 32 to 38°C, for 15 minutes to 24 hours. The reaction is terminated by heating generally at 55 to 70°C, preferably 63 to 67°C for 5 to 10 minutes. Alternatively, the reaction may be terminated by inactivating the restriction enzyme by the addition of an agent such as diethylpyrocarbonate.
  • an agent such as diethylpyrocarbonate.
  • the fragments are incubated with 0.3 to 10 units of T4 DNA ligase at a temperature of 1 to 37°C, preferably 3 to 20°C for 15 minutes to 72 hours, preferably 2 to 20 hours in the presence of 2 to 200 mM, preferably 10 to 40 mM Tris-HCl (pH 6.0 to 9.5, preferably pH 7.0 to 8.0), 2 to 20 mM, preferably 5 to 10 mM MgCl 2 , 0.1 to 10 mM, preferably 0.5 to 2 mM ATP and 1 to 50 mM, preferably 3 to 20 mM dithiothreitol.
  • T4 DNA ligase at a temperature of 1 to 37°C, preferably 3 to 20°C for 15 minutes to 72 hours, preferably 2 to 20 hours in the presence of 2 to 200 mM, preferably 10 to 40 mM Tris-HCl (pH 6.0 to 9.5, preferably pH 7.0 to 8.0), 2 to 20 mM, preferably 5 to 10
  • plasmid of pBR 325 and P 1 fragment i.e., DNA transducing vector is constructed.
  • the P 1 fragment may be inserted into pBR 325 after digestion with Eco RI and Barn HI or Eco RI and Sal I. Moreover, the P 1 fragment can be inserted after the digestion of pBR 325 with Pst I.
  • any plasmid vector may be employed in the present invention in which the P 1 promoter is inserted and shows promoter activity.
  • a plasmid vector having restriction sites digestable with at least one member selected from (1) EcoRI, (2) Hind III and (3) a combination of Eco RI or Hind III and Bam HI, Sal I or Pst I is used.
  • pBR 325, pBR 322, pBR 327, pBR 328 and. p GA 22 are examples.
  • Vector pBR 322 is described in Gene 2, 95 (1977) (F.
  • Bolivar and pBR 327 and pBR 328 are described in Gene 9, 287 (1980) (X. Soberon et al). These plasmid vectors are digested with Eco RI or Hind III and the P 1 fragment is inserted into the digested vector to obtain a DNA transducing vector carrying the P 1 promoter of ribosomal RNA.
  • the pGA 22 vector is described in J. Bacteriol. 140, 400 (1979) (G. An et al).
  • a DNA vector which carries the P 1 fragment and ampicillin resistance gene or a DNA vector which carries the P 1 fragment, ampicillin resistance gene and kanamycin resistance gene is constructed by inserting the P 1 fragment into pGA 22 after digestion with Eco RI and Hind III.
  • the P 1 fragment can be directly inserted into a plasmid or phage DNA which has a structural gene coding for growth hormone, insulin, interferon and the like. Furthermore, a DNA transducing vector carrying the P 1 fragment can be inserted into such plasmid or phage DNA.
  • Such recombinant DNA can be constructed by making a P 1 fragment, a plasmid vector or phage vector and a structural gene independently and combining them in one step or successive steps.
  • T4DNA ligase In the combining process for the construction of the recombinant DNA, T4DNA ligase is used.
  • recombinant DNA is a recombinant of the P 1 fragment and a phage vector containing a gene for ⁇ -galactosidase.
  • the P 1 promoter derived from ribosomal RNA is effective for the expression of ⁇ -galactosidase and the promoter activity is more than two times stronger than that of a lactose promoter.
  • a process for constructing such recombinant DNA is as follows.
  • a phage DNA, ⁇ 459 DNA which is a source of the vector and a phage DNA, XRS 205-7 DNA which is a source of the vector and the structural gene, and the P 1 fragment are used.
  • the X459 DNA and ⁇ RS 205-7 DNA are digested with Eco RI, Hind III and Sal I.
  • the restriction enzyme map is illustrated in Flow Sheet IV wherein the length of Xphage DNA is illustrated as 100% (1% of ⁇ phage DNA corresponds to about 480 base pairs) and the black band means deletion of DNA.
  • lac Z means a gene for ⁇ -galactosidase
  • Trp A means a gene for tryptophane synthetase A protein
  • Trp B means a gene for tryptophane synthetase B protein
  • lac Z + and Trp A + mean the presence of the complete structural genes thereof
  • Trp B means the presence of a part of the structural gene thereof.
  • Trp A and lac Z that is, terminator
  • W 205 (see Flow Sheet IV) is one of the Trp - Lac fusions [Mitchell et al., J. Mol Biol., 93, 331 (1975)].
  • the L-fragment carrying Lac Z and Trp A which is obtained by the digestion of ⁇ RS 205-7 DNA with Hind I II, the R-fragment obtained by the digestion of X459 DNA with Eco RI and the P 1 fragment prepared as above are combined with T4 DNA ligase to make a recombinant DNA of "L-fragment (Lac Z + , Trp A + )"- P 1 fragment - "R-fragment".
  • a strain of E. coli is transformed with the recombinant DNA and the transformant is used for the production of ⁇ -galactosidase.
  • the amount of ⁇ -galactosidase produced by the transformant is two or more times that of an E. coli strain containing the lactose promoter. This fact illustrates the superiority of the P 1 fragment as a promoter.
  • a DNA transducing vector carrying the P 1 fragment is constructed as follows:
  • a transducing phage DNA carrying the P 1 promoter of ribosomal RNA gene-E, ⁇ met A 20 DNA is prepared according to the method described in G. Zubay et al., The Lactose Operon, J. R. Beckwith eds (New York: Cold Spring Harbor Laboratory) p. 375 - 391; Yamamoto et al; FEBS Letters, 72, 256 (1976) ; Flow Sheet I.
  • the ⁇ met A 20 DNA is digested with a restriction enzyme Eco RI (product of Bethesda Research Laboratories Co., (hereinafter referred to as "B.R.
  • the digest is subjected to purification by agarose gel electrophoresis to obtain a DNA fragment with 1,370 base pairs.
  • the DNA fragment is digested with a restriction enzyme Hind II (product of B.R.L.) and a restriction enzyme Hind III (product of B.R.L.) and the digest is again subjected to purification by agarose gel electrophoresis to obtain a P1 fragment with 500 base pairs.
  • Flow Sheet I shows a restriction map around the P 1 and P 2 promoters of ribosomal RNA on the phage Xmet A 20 DNA and the size of each fragment.
  • Hind III linker (CCAAGCTTGG), product of Collaborative Research Inc.] is phosphorylated with ATP and polynucleotide kinase according to a conventional method of phosphorylation [Nucleic Acid Res., 5, 4479 (1978)] to obtain phosphorylated Hind III linker.
  • T4 DNA ligase is a product of New England Biolabo Co.
  • the reaction is terminated by heating at 65°C for 5 minutes and the reaction solution is digested with 4 units of Hind III and 4 units of Eco RI at 37°C for 4 hours. All of the reaction mixture (40 ⁇ l) is charged on a column packed with 2 ml of Sephadex G-100 and elution is carried out with a mixture of 20 mM Tris-HCl (pH 7.8), 50 mM NaCl and 10 mM disodium ethylenediaminetetraacetate (EDTA) (concentrations given are the final) to remove extra Hind III linker.
  • EDTA disodium ethylenediaminetetraacetate
  • the plasmid vector pBR 325 [F. Bolivar, Gene 4, 121 (1978)] prepared according to the method described in P. Guerry et al., J. Bacteriol. 116, 1064 (1973) is used.
  • 5 ⁇ g of pBR 325 is digested with 10 units of Hind III and 10 units of Eco RI at 37°C for 2 hours in the presence of 20 mM Tris-HCl (pH 7.6), 60 mM NaCl and 7 mM MgCl 2 and the reaction is terminated by heating at 65°C for 5 minutes.
  • 0.1 pg of the pBR 325 digested with Hind III and Eco RI and 5 ⁇ l of a solution containing 0.5 ⁇ g of the P 1 fragment having the Hind III end obtained as above are mixed and 10 pl of a solution of 20 mM Tris-HCl (pH 7.6), 10 mM MgCl 2 , 10 mM dithiothreitol and 2 mM ATP are added to make up 20 ⁇ l of a solution.
  • telomere sequence is named pKS 1003.
  • a microorganism is prepared wherein a recombinant plasmid carrying the P 1 fragment is introduced.
  • the recombinant plasmid carrying the P 1 fragment obtained in Example 1, i.e. DNA transducing vector is introduced into a microorganism in the following manner.
  • Escherichia coli C 600 SF 8 [Cameron et al, Proc. Natl. Acad. Sci., 72, 3416 (1975)] is used as a recipient strain and transformation is performed according to S. N. Cohen's method [Proc. Natl. Acad. Sci., 69, 2110 (1972)].
  • transformants of E . coli C 600 SF 8 which carry plasmid pKS 1003 are obtained by selecting a tetracycline resistant (TC R ) and a chloramphenicol sensitive (CM S ) strain, because the parent plasmid pBR 325 carries genes for AP R , TC R and CM R and the recombinant plasmid carrying the P 1 promoter derived from ribosomal RNA carries genes for AP R , TC R and CM S .
  • DNAs are prepared from E. coli C 600 SF 8 (APR, TC R , CM s ) by a conventional method and digested with Eco RI and Hind III. The presence of the P 1 fragment (500 base pairs) is confirmed by agarose gel electrophoresis.
  • the microorganism strain containing the DNA transducing vector obtained in this Example is named E scherichia coli I-001.
  • the strain has been deposited with the American Type Culture Collection, U.S.A. and has been accorded accession number ATCC 31698.
  • a recombinant DNA carrying the P 1 fragment is constructed as follows with reference to the following Flow Sheet III illustrating the processes for constructing the recombinant DNA, and the following Flow Sheet IV illustrating the restriction maps of X459 DNA, ⁇ RS 205-7 DNA and the recombinant DNA carrying the P 1 fragment.
  • a recombinant DNA wherein the L-fragment of ⁇ RS205-7 DNA, P 1 fragment and R-fragment of ⁇ 459 DNA combined in this order is obtained.
  • the recombinant DNA carries Lac Z and Trp A on the L-fragment.
  • Detection of the Lac Z gene ( ⁇ -galactosicase gene) on the recombinant DNA is performed by subjecting the recombinant DNA to transfection using E. coli LacO - Z - , a mutant strain which has no operator and structural gene for ⁇ -galactosidase, as an indicator strain.
  • the phage containing the recombinant DNA is detected as a blue plaque on a bouillon agar plate containing 5-bromo-4- hloro-3-indoyl- ⁇ -D-galactoside.
  • phage containing the recombinant DNA is named ⁇ P 1 -Lac 1.
  • the presence of the P 1 promoter on ⁇ P 1 -Lac 1 DNA is confirmed by the fact that the P 1 fragment (500 base pairs) is generated when ⁇ P 1 -Lac 1 DNA is digested with Hind III and Eco RI.
  • a microorganism strain is prepared wherein a recombinant DNA is introduced.
  • Escherichia coli strain wherein the gene for ⁇ -galactosidase is deleted, i.e. Escherichia coli B 2550 Lac O - Z - Y c Trp - [Y means constitutive property for permease and Trp means requirement for tryptophan] is lysogenized with the recombinant phage obtained in Example 3.
  • a strain lysogenized with X plac 5 which is a phage carrying the lactose operon of E. coli [K. Ippen et al., J. Bact., 108, 5 - 9 (1971)] is used.
  • the lysogenic strains are inoculated into 2 ml of LB broth wherein 10 g of bactotryptone, 5 g of yeast extract and 5 g of NaCl are dissolved in 1 l of water and the solution is adjusted to pH 7.0 with NaOH and 2 ml of LB broth containing 1 mM isopropylthiogalactoside (IPTG) (referred to as "LB + IPTG broth” hereinafter), and cultivated at 30°C for 18 hours. Then, 0.25 ml of the cultures are inoculated into 5 ml of the same brothes and the cultivation is carried out at 30°C for 3 hours.
  • IPTG isopropylthiogalactoside
  • the lysogenic strain with ⁇ P 1 -Lac 1 is named Escherichia coli I-002.
  • the strain has been deposited with the American Type Culture Collection, U.S.A. and accorded accession number ATCC 31699.
  • a medium 0.4% Glucose is added to a minimum medium which consists of 6 g/l Na 2 HPO 4 , 3 g/l KH 2 PO 4 , 0.5 g/l NaCl, 1 g/l NH 4 Cl and 0.1 mM CaCl 2 (the same as hereinafter).
  • B medium A minimum medium containing 0.4% glucose and 0.4% casamino acid.
  • C medium An LB broth containing 0.4% glucose. Culturing is carried out at 30°C.
  • Fig. 1 The results show the production of ⁇ -galactosidase by the lysogenic strain with ⁇ P 1 -Lac 1 increases greatly in a medium wherein the strain grows rapidly which coincides with the information that ribosomal RNA increases in proportion to the growth of the strain [N. O. Kjeldgaard: J. Mol. Biol, 6, 341 (1963)].

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EP81107594A 1980-09-24 1981-09-23 Vecteur de transduction de DNA et micro-organisme le contenant Withdrawn EP0048497A3 (fr)

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JP55131457A JPS5756495A (en) 1980-09-24 1980-09-24 Novel dna-introduction vector and recombinant dna
JP131457/80 1980-09-24

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EP0048497A2 true EP0048497A2 (fr) 1982-03-31
EP0048497A3 EP0048497A3 (fr) 1983-01-19

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2573773A1 (fr) * 1984-11-26 1986-05-30 Richter Gedeon Vegyeszet Vecteurs de replication et leur procede de production
US4716112A (en) * 1982-09-16 1987-12-29 Biogen N.V. Vectors for increased expression of cloned genes

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS59116291A (ja) * 1982-12-06 1984-07-05 Fujisawa Pharmaceut Co Ltd ７−置換−３−セフエム−４−カルボン酸エステルおよびその製法
JPS59162874A (ja) * 1983-03-08 1984-09-13 Rikagaku Kenkyusho 微生物の培養方法
JPS60126077A (ja) * 1983-12-09 1985-07-05 Rikagaku Kenkyusho 微生物の培養方法
JPS6062984A (ja) * 1983-09-19 1985-04-11 Wakamoto Pharmaceut Co Ltd 耐熱性β−ガラクトシダ−ゼ遺伝子を有する新規な大腸菌及び耐熱性β−ガラクトシダ−ゼの製造法
JPS63202385A (ja) * 1987-02-19 1988-08-22 Mitsui Toatsu Chem Inc 外来遺伝子の発現制御方法及び該方法を用いた外来遺伝子産物の生産方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol.91, no.7, August 13, 1979, page 329, abstract no.52485k, Columbus, Ohio (US) *
CHEMICAL ABSTRACTS, vol.91, no.7, August 13, 1979, page 330, abstract no.52486m, Columbus, Ohio (US) *
CHEMICAL ABSTRACTS, vol.92, no.5, February 4, 1980, page 287, abstract no.36206k, Columbus, Ohio (US) *
CHEMICAL ABSTRACTS, vol.93, no.23, December 8, 1980, page 277, abstract no.217838j, Columbus, Ohio (US) *
CHEMICAL ABSTRACTS, vol.95, no.19, November 09, 1981, page 394, abstract no.165394f, Columbus, Ohio (US) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4716112A (en) * 1982-09-16 1987-12-29 Biogen N.V. Vectors for increased expression of cloned genes
FR2573773A1 (fr) * 1984-11-26 1986-05-30 Richter Gedeon Vegyeszet Vecteurs de replication et leur procede de production

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EP0048497A3 (fr) 1983-01-19

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